Histocompatibility of Light-Curing Composites Used in Pediatric Dentistry in Human Oral Fibroblast Cells.

Statement of the Problem: Tooth-colored composites are used to repair caries lesions and other dental defects, particularly in anterior regions in children. Although a wide range of composites is using, little attention has been paid to the important indicators such as biological profiles or products released from these materials. Purpose: The current study aimed to compare the histocompatibility and cytotoxicity of light-curing resin used to repair children's teeth with different brands (3M, DenFil, and Opallis) in curing times of 20 and 40 seconds in human oral fibroblast cells (HGF1). Materials and Method: In this in vitro study, Three types of flow composites (3M, Opallis, and DenFil), all at A2 shade, were used. The composites were at 4×2mm with separate exposure times of 20 and 40 seconds. MTT test was used to determine the cytotoxicity of composites on oral fibroblast cells. This test is based on the conversion of tetrazolium bromide to a purple compound known as formazan that its color intensity can be evaluated using the ELISA. The higher intensity of the color reveals the higher survival rate of the mitochondria, which indicates less toxicity. One-way variance analysis and unpaired t-test were used to compare the cytotoxicity of each brand in two conditions of 20 and 40 seconds of curing. Statistical significance was considered when p< 0.05. Results: 3M and Opallis composites were significantly reduced vitality of cells compared to control group in both 20s and 40s curing status. While DenFil brand did not show marked cytotoxicity. In each brand, there are no significant deference between 20s and 40s curing times. Conclusion: Histocompatibility depends on the type of composite resin. In the current study, DenFil brand showed the highest histocompatibility, followed by 3M and Opallis.

A comparison of microleakage in self-etch fissure sealants and conventional fissure sealants with total-etch or self-etch adhesive systems.

OBJECTIVE: The present study was conducted to compare microleakage in self-etching fissure sealants and conventional fissure sealants with total-etch or self-etch adhesive systems. SETTINGS AND DESIGN: This experimental in vitro study was conducted on 60 healthy third molars extracted from humans. The first group received Acid etch + Clinpro sealant, the second group received Acid etch + Single bond 2 + Clinpro sealant, the third group received Single bond universal (self-etching bonding) + Clinpro sealant, and the fourth group received prevent seal self-etching sealant. MATERIALS AND METHODS: An incision was made on the teeth after they were immersed in methylene blue 5%. The samples were then examined under a stereomicroscope and the dye penetration rate was measured based on the Williams and Winter criteria. STATISTICAL ANALYSIS USED: The Kruskal-Wallis and Mann-Whitney tests were used for data analysis in SPSS-18 (P < 0.05). RESULTS: Group 1 which was treated with the conventional technique (acid + fissure sealant) had the highest rate of microleakage compared to Groups 2 and 3 (P < 0.001). CONCLUSION: The results showed that the use of bonding results in a significant reduction in the microleakage of fissure sealants. The microleakage caused when using self-etch fissure sealant was not different from that caused by the use of the conventional method.

Role of Anatomic and Salivary Factors in Dental Calculus Formation in Primary and Mixed Dentition Stages.

PURPOSE: Clinical experience shows that formation of calculus is a very rare phenomenon in primary teeth, but it increases as the permanent teeth erupt. The purpose of this study was to assess the relationship between dental calculus, dental anatomy, and salivary factors in primary and mixed dentition stages. METHODS: A cross-sectional study was carried out to determine the buccolingual dimensions of the most concave and the most convex surfaces of the lingual aspect of mandibular central incisor crowns in a sample group of 120 three- to five-old children and 120 eight- to 10-year old children. Saliva samples were collected from 20 in each group. Data were analyzed using t tests. RESULTS: Significant differences were found between the ratio of the buccolingual dimensions of the most convex to the most concave areas of the lingual surfaces in primary and permanent incisors (P=0.028). Saliva analysis revealed significant differences in total protein (P=0.002), sodium (P=0.037), bicarbonate (P=0.003), and ammonia (P=0.025) between the two age groups. CONCLUSIONS: Anatomic and salivary factors may be important reasons for the differences in calculus formation.

Bond Strength of 5(th), 6(th) and 7(th) Generation Bonding Agents to Intracanal Dentin of Primary Teeth.

OBJECTIVES: This in-vitro study sought to assess the push-out bond strength of a total etch and 2 self-etch bonding systems to intracanal dentin of primary anterior teeth (PAT). MATERIALS AND METHODS: Thirty-six primary anterior teeth were randomly divided into 3 groups of 5(th) generation (Single Bond 2), 6(th) generation (Clearfil SE) and 7(th) generation (Single Bond Universal) bonding agents. The canal orifice was restored with composite resin and the push-out test was carried out to assess the bond strength. After applying the push-out load, specimens were evaluated under a light microscope at 40X magnification. One-way ANOVA and log-rank test on Kaplan-Meier curves were applied for the comparison of bond strength among the 3 groups. RESULTS: The mean± standard deviation (SD) bond strength was 13.6±5.33 MPa for Single Bond 2, 13.85±5.86 MPa for Clearfil SE and 12.28±5.24 MPa for Single Bond Universal. The differences in bond strength among the 3 groups were not statistically significant (P>0.05). CONCLUSION: All three bonding agents are recommended for use with composite posts in PAT. However, due to high technical sensitivity of the Total Etch system, single or two-step self etch systems may be preferred for uncooperative children.

Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma.

PURPOSE: Infusion of interleukin-12 (IL12) can mediate antitumor immunity in animal models, yet its systemic administration to patients with cancer results in minimal efficacy and severe toxicity. Here, we evaluated the antitumor activity of adoptively transferred human tumor-infiltrating lymphocytes (TILs) genetically engineered to secrete single-chain IL12 selectively at the tumor site. EXPERIMENTAL DESIGN: Thirty-three patients with metastatic melanoma were treated in a cell dose-escalation trial of autologous TILs transduced with a gene encoding a single-chain IL12 driven by a nuclear factor of the activated T cells promoter (NFAT.IL12). No IL2 was administered. RESULTS: The administration of 0.001 to 0.1 × 10(9) NFAT.IL12-transduced TILs to 17 patients resulted in a single, objective response (5.9%). However, at doses between 0.3 and 3 × 10(9) cells, 10 of 16 patients (63%) exhibited objective clinical responses. The responses tended to be short, and the administered IL12-producing cells rarely persisted at 1 month. Increasing cell doses were associated with high serum levels of IL12 and IFNγ as well as clinical toxicities, including liver dysfunction, high fevers, and sporadic life-threatening hemodynamic instability. CONCLUSIONS: In this first-in-man trial, administration of TILs transduced with an inducible IL12 gene mediated tumor responses in the absence of IL2 administration using cell doses 10- to 100-fold lower than conventional TILs. However, due to toxicities, likely attributable to the secreted IL12, further refinement will be necessary before this approach can be safely used in the treatment of cancer patients.

Clinical-scale selection and viral transduction of human naïve and central memory CD8+ T cells for adoptive cell therapy of cancer patients.

The adoptive transfer of lymphocytes genetically engineered to express tumor-specific antigen receptors is a potent strategy to treat cancer patients. T lymphocyte subsets, such as naïve or central memory T cells, selected in vitro prior to genetic engineering have been extensively investigated in preclinical mouse models, where they demonstrated improved therapeutic efficacy. However, so far, this is challenging to realize in the clinical setting, since good manufacturing practices (GMP) procedures for complex cell sorting and genetic manipulation are limited. To be able to directly compare the immunological attributes and therapeutic efficacy of naïve (T(N)) and central memory (T(CM)) CD8(+) T cells, we investigated clinical-scale procedures for their parallel selection and in vitro manipulation. We also evaluated currently available GMP-grade reagents for stimulation of T cell subsets, including a new type of anti-CD3/anti-CD28 nanomatrix. An optimized protocol was established for the isolation of both CD8(+) T(N) cells (CD4(-)CD62L(+)CD45RA(+)) and CD8(+) T(CM) (CD4(-)CD62L(+)CD45RA(-)) from a single patient. The highly enriched T cell subsets can be efficiently transduced and expanded to large cell numbers, sufficient for clinical applications and equivalent to or better than current cell and gene therapy approaches with unselected lymphocyte populations. The GMP protocols for selection of T(N) and T(CM) we reported here will be the basis for clinical trials analyzing safety, in vivo persistence and clinical efficacy in cancer patients and will help to generate a more reliable and efficacious cellular product.

Evaluation of γ-retroviral vectors that mediate the inducible expression of IL-12 for clinical application.

The clinical application of interleukin-12 (IL-12) has been hindered by the toxicity associated with its systemic administration. To potentially overcome this problem, we developed a promoter designed to direct IL-12 expression within the tumor environment using an inducible composite promoter containing binding motifs for the nuclear factor of activated T cells (NFAT) linked to a minimal IL-2 promoter. In this study, the NFAT promoter was coupled to a single-chain human IL-12 gene and inserted into 2 γ-retroviral self-inactivating vectors (SERS.NFAT.hIL12 and SERS.NFAT.hIL12.PA2) and 1 γ-retroviral vector (MSGV1.NFAT.hIL.12 PA2). Peripheral blood lymphocytes (PBLs) were double transduced with an antigen-specific T-cell receptor and the 3 NFAT.hIL12 vectors. Evaluation of inducible IL-12 expression, transduction efficiency, and vector production considerations led to the choice of the MSGV1.NFAT.hIL12.PA2 vector for clinical application. MSGV1.NFAT.hIL12.PA2 PG13 retroviral vector producer cell clones were screened by transduction of tumor antigen-specific PBLs. On the basis of expression studies in PBL, clone D3 was chosen to produce clinical-grade viral vector supernatant and was demonstrated to efficiently transduce young tumor-infiltrating lymphocytes (TIL). The vector-transduced young TIL with known tumor recognition demonstrated specific inducible IL-12 production after coculture with HLA-matched tumor targets and had augmented effector function as demonstrated by increased IFN-γ secretion. These results support the clinical application of adoptive transfer of young TIL engineered with the NFAT.hIL12 vector as a new approach for cancer immunotherapy.

Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1.

PURPOSE: Adoptive immunotherapy using tumor-infiltrating lymphocytes represents an effective cancer treatment for patients with metastatic melanoma. The NY-ESO-1 cancer/testis antigen, which is expressed in 80% of patients with synovial cell sarcoma and approximately 25% of patients with melanoma and common epithelial tumors, represents an attractive target for immune-based therapies. The current trial was carried out to evaluate the ability of adoptively transferred autologous T cells transduced with a T-cell receptor (TCR) directed against NY-ESO-1 to mediate tumor regression in patients with metastatic melanoma and synovial cell sarcoma. PATIENTS AND METHODS: A clinical trial was performed in patients with metastatic melanoma or metastatic synovial cell sarcoma refractory to all standard treatments. Patients with NY-ESO-1-positive tumors were treated with autologous TCR-transduced T cells plus 720,000 iU/kg of interleukin-2 to tolerance after preparative chemotherapy. Objective clinical responses were evaluated using Response Evaluation Criteria in Solid Tumors (RECIST). RESULTS: Objective clinical responses were observed in four of six patients with synovial cell sarcoma and five of 11 patients with melanoma bearing tumors expressing NY-ESO-1. Two of 11 patients with melanoma demonstrated complete regressions that persisted after 1 year. A partial response lasting 18 months was observed in one patient with synovial cell sarcoma. CONCLUSION: These observations indicate that TCR-based gene therapies directed against NY-ESO-1 represent a new and effective therapeutic approach for patients with melanoma and synovial cell sarcoma. To our knowledge, this represents the first demonstration of the successful treatment of a nonmelanoma tumor using TCR-transduced T cells.

Relationship of p53 overexpression on cancers and recognition by anti-p53 T cell receptor-transduced T cells.

Tumor suppressor p53 is reported to be an attractive immunotherapy target because it is mutated in approximately half of human cancers, resulting in inactivation and often an accumulation of the protein in the tumor cells. Only low amounts of protein are detectable in normal tissues. The differential display of antigen in normal versus tumor tissues has been reported to create an opportunity to target p53 by immunotherapy. We sought to determine the relationship between p53 expression and its recognition by cognate T cells in human tumors including common epithelial malignancies. Inasmuch as nonsense or missense p53 mutations may disrupt processing and presentation, we studied tumors with either identified wild-type or mutated p53, based on our gene-sequencing studies or published data. T cells transduced with a high-affinity, p53(264-272)-reactive T cell receptor (TCR) derived from HLA-A2.1 transgenic mice recognized a wide panel of human tumor lines. There was no significant correlation between p53 expression in tumors and recognition by the anti-p53 TCR-transduced T cells. This conclusion was based on the study of 48 cell lines and is in contrast to several prior studies that used only a limited number of selected cell lines. A panel of normal cells was evaluated for recognition, and some of these populations were capable of stimulating anti-p53 T cells, albeit at low levels. These studies raise doubts concerning the suitability of targeting p53 in the immunotherapy of cancer patients.

Cancer regression in patients after transfer of genetically engineered lymphocytes.

Through the adoptive transfer of lymphocytes after host immunodepletion, it is possible to mediate objective cancer regression in human patients with metastatic melanoma. However, the generation of tumor-specific T cells in this mode of immunotherapy is often limiting. Here we report the ability to specifically confer tumor recognition by autologous lymphocytes from peripheral blood by using a retrovirus that encodes a T cell receptor. Adoptive transfer of these transduced cells in 15 patients resulted in durable engraftment at levels exceeding 10% of peripheral blood lymphocytes for at least 2 months after the infusion. We observed high sustained levels of circulating, engineered cells at 1 year after infusion in two patients who both demonstrated objective regression of metastatic melanoma lesions. This study suggests the therapeutic potential of genetically engineered cells for the biologic therapy of cancer.

A phase I study of nonmyeloablative chemotherapy and adoptive transfer of autologous tumor antigen-specific T lymphocytes in patients with metastatic melanoma.

This report describes a phase I clinical trial using nonmyeloablative, lympho-depleting chemotherapy in combination with adoptive immunotherapy in patients with metastatic melanoma. The chemotherapy-conditioning schedule that induced transient lymphopenia consisted of cyclophosphamide (30 or 60 mg/kg per day for 2 days) followed by fludarabine (25 mg/m(2) per day for 5 days). Immunotherapy for all patients consisted of in vitro expanded, tumor-reactive, autologous T-cell clones selected for high avidity recognition of melanoma antigens. Cohorts of three to six patients each received either no interleukin (IL)-2, low-dose IL-2 (72,000 IU/kg intravenously three times a day to a maximum of 15 doses), or high-dose IL-2 (720,000 IU/kg intravenously three times a day for a maximum of 12 doses). The toxicities associated with this treatment were transient and included neutropenia and thrombocytopenia that resolved in all patients. High dose intravenous IL-2 was better tolerated by patients after chemotherapy than during previous immunotherapy cycles without chemotherapy. No patient exhibited an objective clinical response to treatment, although five patients demonstrated mixed responses or transient shrinkage of metastatic deposits. This study established a nonmyeloablative-conditioning regimen that could be safely administered in conjunction with adoptive T-cell transfer and IL-2 in patients with metastatic melanoma.